EP3806832A1 - Electrospun fibers for a local release of an anti-inflammatory drug and a promyelinating drug - Google Patents

Electrospun fibers for a local release of an anti-inflammatory drug and a promyelinating drug

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Publication number
EP3806832A1
EP3806832A1 EP18752288.3A EP18752288A EP3806832A1 EP 3806832 A1 EP3806832 A1 EP 3806832A1 EP 18752288 A EP18752288 A EP 18752288A EP 3806832 A1 EP3806832 A1 EP 3806832A1
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EP
European Patent Office
Prior art keywords
agent
fibers
promyelinating
inflammatory
electrospun
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18752288.3A
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German (de)
English (en)
French (fr)
Inventor
Laura Calza'
Luciana Giardino
Maria Letizia Focarete
Chiara Gualandi
Maria-Laura BOLOGNESI
Nadia PASSERINI
Giampiero PAGLIUCA
Teresa GAZZOTTI
Elisa ZIRONI
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Universita di Bologna
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Universita di Bologna
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Publication of EP3806832A1 publication Critical patent/EP3806832A1/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/60—Salicylic acid; Derivatives thereof
    • A61K31/603—Salicylic acid; Derivatives thereof having further aromatic rings, e.g. diflunisal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • Fig. 11 shows the inhibition of GABA release from synaptosomes obtained by the spinal cord of rat implanted with the invention, in comparison with the polymer, alone, to prove the specificity on glutamate release
  • Fig. 12 shows the cell composition of the cellular infiltrate in the spinal cord of rat implanted with the invention, in comparison with the polymer, alone, proving the increase of fagocytes (CD11+) and promyelinating cells (MBP+).
  • Fig. 14 shows the best polymer chemistry for the drug release profile required for the purpose of the invention
  • said fibers have a diameter between 50 and 5000 nm.
  • the voltage used in the electrospinning steps of the process is between 15 and 20 kV and/or a flow rate between 0.5 and 2.5 ml/h.
  • the blend solution comprises a promyelinating agent in a concentration between 0.1 and 2% weight/weight (w/w) and/or the anti-inflammatory agent in a concentration between 1 and 10% w/.
  • DMF Dimethylformamide
  • DMC dimethyl carbonate
  • MetalOH Methanol
  • DMF dichloromethane
  • MeOH Methanol
  • T3 3,3,5-Triiodo-L-thyronine sodium salt >95% (HPLC) was purchased from Sigma Aldrich. Ibuprofen was purchased from Sigma Aldrich.
  • Ibuprofen was analyzed by HPLC system consisted of two mobile phase delivery pumps (LC-lOADvp, Shimadzu, Japan) and a UV-vis detector (SPD-lOAvp, Shimadzu, Japan).
  • An autosampler (SIL-20A, Shimadzu, Japan) was used to inject samples (20 m ⁇ ) onto a Kinetex Cl 8 (150 mm x 4.60 mm x 5 pm) column
  • the mobile phase consisted of NH4H2P04 (0.02 M with pH adjusted at 3.0 using ortho phosphoric acid) and CH3CN (43:57, V/V).
  • the mobile phase was filtered through a 0.22-pm Sartorius filter before use.
  • the flow rate was 1 ml/min and the detection wavelength was set at 220 nm.
  • the retention time of IBU was 5.2 minutes.
  • Linearity was assessed by analyzing IBU at seven different concentrations within 0.05-20 pg/ml of the nominal standard concentration.
  • the linearity of the proposed method was evaluated by using calibration curve to calculate coefficient of correlation, slope, and intercept values.
  • LOD is the lowest amount of analyte in a sample that can be detected but not necessarily quantified.
  • LOQ is the lowest amount of analyte in a sample that may be determined with acceptable accuracy and precision.
  • the values corresponding to LOD and LOQ were estimated as IBU concentrations that give rise to peaks whose heights correspond to 3 and 10 times the background noise.
  • the mobile phase consisted of a mixture of water/methanol (90/10, v/v) containing 0,1 % of formic acid (solvent A) and methanol containing 0,1% of formic acid (solvent B).
  • the gradient (constant flow rate at 0.3 mL/min) started with 70% A and 30% B, then followed by a gradient of solvent B (min 2-75%, min 2.5-85%, min 2.6- 95%, min 2.8-10%, min 3.5-75%, min 4-60%, min 5-30%).
  • the chromatographer was interfaced with a Waters Quattro Premier XE tandem mass spectrometer, equipped with an ESCi multi-mode ionization source (Waters Corporation, Milford, MA, USA) and operating in negative electrospray ionization (ESI +) mode. Analysis was performed in MRM (multiple reaction monitoring) mode, following two transitions for T3 (in brackets the relative optimized values of cone voltage and collision energy): 65l.45>605.4 (33 v, 35 eV) and 65l.45>478.7 (35 V, 30 eV).
  • capillary voltage was set at 3.00 kV and cone voltage at 30 V, while source and desolvation temperatures were
  • Nitrogen flow was set at 50 L/h on the cone and 500 L/h for desolvation; argon was used as collision gas at 0.35 mL/min.
  • Data acquisition and processing were performed using MassLynx 4.1 software (Waters Corporation, Milford, MA, USA).
  • Matrix- matched calibration curve was prepared using DMEM cell colture medium, that was spiked at 5 different concentrations of T3 (0, 10, 50, 100, 200 ng/mL).
  • the murine macrophage cell line RAW 264.7 was purchased from the American Type Culture Collection (ATCC® TIB-71TM). Cells were grown at 37 °C in DMEM high glucose medium (Thermo Fisher Scientific) supplemented with 10% heat- inactivated fetal bovine serum (FBS - Thermo Fisher Scientific), 1% penicillin/streptomycin (100 U ml-l/lOO pg ml-1) (Thermo Fisher Scientific), in a humidified incubator of 5% C02. When 70-80% confluency was reached, cells were detached mechanically with the scraper and subcultured in 75 cm2 flasks. Treatments
  • RNA isolation and semi-quantitative real-time polymerase chain reaction qPCR
  • Thermal profile of PCR reactions consisted first of a denaturation step (95°C, 2 min) and 40 cycles of amplification (95°C for 15 s and 60°C for 60 s). At the end of the amplification cycles, the melting curve of amplified products was performed according to the following temperature/time scheme: heating from 55°C to 95°C with a temperature increase of 0.5°C/s. Primer efficiency values for all primers were 95-102%. Relative quantification of the target genes expression was calculated using the comparative threshold method The primers used are also disclosed in Giardino L. et al. Glia 2006 Sep;(64(9): 1573-89. doi: 10.1002/glia.23025.
  • CD-Sprague Dawley (Charles River) female rats of 200-250 gr were used for this study. All animals were housed in pairs with standard bedding and diet. One week before surgery all animals were handled and accustomed to bladder manipulation. Surgery and animal care
  • mice underwent a contusive spinal lesion at the thoracic level (T9). Briefly, rats were anesthetized with isoflurane (1-3%) in 02 and treated before surgery with enrofloxacin and tramadol (4 mg/kg, s.c.). Laminectomy was performed at the T8- T10 vertebral level under aseptic conditions. The dura was carefully open and spinal cord was exposed.
  • Systemic treatment was administered daily via sub-cutaneous injections starting after two hours after spinal lesion and for the successive 10 days. Animals were treated with 60 mg/kg of ibuprofen once a day and 10 ug/kg of T3 twice a day (Fernandez et al., 2004; D’intino et al, 201 1; Dell’Acqua et al, 2012).
  • Implantation of ibuprofen and T3 conjugated scaffolds was performed after spinal cord lesion. Electrospun was placed upon the spinal cord, bent over it and then fixed with BioGlue (CryoLife) at the spinal process of adjacent vertebrae.
  • Gait analysis was performed on 11 different parameters, divided in 4 categories: Spatial Parameters (Print Area, Max Contact Area, Base of Support); Kinetic Parameters (Stand Time, Swing Time, Swing Speed, Single Stance); Comparative Parameters (Stride Length, Step Cycle); Coordination Parameters (Duty Cicle, Step Sequence Regularity Index). All parameters were analyzed for both hind paws and front paws (represented as mean of right paw and left paw).
  • RNA isolation was performed using RNeasy Microarray Tissue Mini Kit (Qiagen). Total RNA was eluted in RNase Free Water and using a spectrophotometer (Nanodrop 2000, Thermo Scientific), absorbance values were measured and purity (A260/A280) evaluated. RNAs were pooled for each group to obtain a total amount of RNA of 0.5 pg. Genomic DNA was digested using GE Buffer (Qiagen) for 5 minutes at 42°C and then retrotranscribed using the RT2 First Strand Kit (Qiagen) and thermal cycler for 15 minutes at 42°C followed by 5 minutes at 92°C for enzyme inhibition. Synaptic Plasticity analysis was performed using Rat Synaptic Plasticity PCR Array (PARN- 126ZD - Qiagen), a specific panel of 84 genes involved in Rat Synaptic Plasticity
  • the dissociation curve was obtained by following a procedure consisting of first incubating samples at 95 °C for lmin to denature the PCR-amplified products, then ramping temperature down to 65 °C and finally increasing temperature from 65 °C to 95 °C at the rate of 0.5 °C/s, continuously collecting fluorescence intensity over the temperature ramp.
  • Analysis of genes expression of the array was performed using the RT2 Profiler PCR Array Data Analysis version 3.5 (SABiosciences), previously expression threshold was set in order to analyze only linear expression and maximum Ct was set at the value of 35. Gene expression was normalized on automatically detected reference genes suggested by the software.
  • tissues were dissociated in single cells with Multi Tissue Dissociation Kit 1 (Miltenyi Biotech) using GentleMACS Dissociators (Miltenyi Biotech) for 45 minutes. Tissue debris were removed using Debris Removal Solution (Miltenyi Biotech) in order to eliminate myelin and cellular debris. Cells were marked with a panel of antibodies (Table %) in order to evaluate inflammation, oligodendrocite population and vitality of cells.
  • MACSQuant Flow Cytometer (Miltenyi Biotech) was used to separate and count cell populations marked with specific antibody. Compensation of instrument channels were performed before sample analysis in order to prevent laser overlay during sample reading.
  • the P2 pellet fraction was resuspended in 5 ml of Krebs solution (mm: NaCl 118.5, KC1 4.7, CaCl2 1.2, KH2P04 1.2, MgSC)4 1.2, NaHC03 25, glucose 10; gassed with 95% 02/5% C02) (Chiodi et al. 2012).
  • synaptosomes were maintained in warm condition (37°C) for 20 min. Thereafter, identical aliquots (0.5 ml) of synaptosomal suspension were distributed on microporous filters, placed at the bottom of a set of parallel superfusion chambers, maintained at 37°C and continuously perfused with aerated (95% 02/5% C02) Krebs solution (0.3 ml/min). After a 30-min wash-out period, nine consecutives 5-min fractions were collected. In details, after the collection of three basal samples, synaptosomes were depolarized with 15 mm K+ for 90 seconds. In each sample, GABA and glutamate levels were simultaneously measured by HPLC coupled with fluorimetric detection.
  • the column was eluted at a flow rate of 0.52 ml/min (Beckman 125 pump; Beckman Instruments Indianapolis, IN, USA) with a mobile phase containing 0.1 m sodium acetate, 10% methanol and 2.2% tetrahydrofuran (pH 6.5). Glutamate and GABA were detected by means of a Jasco fluorescence spectrophotometer FP-2020 Plus (Jasco, Tokyo, Japan). The retention times of glutamate and GABA were ⁇ 3.5 and ⁇ 15.0 min, respectively.
  • GABA and glutamate efflux were expressed as pmol/min/g of protein while K+-evoked GABA and glutamate efflux were expressed as percent increase over the respective spontaneous efflux (calculated by the mean of the two fractions collected prior to the depolarising stimulus). Protein was determined according to Bradford (1976).
  • PLLA a part of fiber, accurately weighted, was dissolved in 2 mL of ACN : DMSO (3:lv/v), warmed up and sonicated for 15 minutes, finally the solutions were centrifuged for 10 minutes; if necessary, dilutions were made to get a concentration in the range of 0.05-20 pg/ml. Then, the IBU content of the solution was assayed by HPLC, as reported in the previously Section.
  • Wa is the actual drug content and Wt is the theoretical drug content.
  • the withdrawn fluid samples were then acidificated with HC1 cone, filtered (0.4 um NY filter), diluted if necessary, then the drug content was analyzed by HPLC as described above. The release tests were performed at least in triplicate and the mean ⁇ SD was reported.
  • the standard solution (10 pg/mL) was injected using at different mobile phase ratios. Increasing the percentage of organic phase, the retention time shifted from 7.1 to 5.2 minutes; in all cases the IBU peak is baseline resolved and showed good symmetry, the optimal condition was obtained using the 57 :43 v/v ratio.
  • the method was also evaluated for the determination of IBU in presence of T3 (hormone thyroid) and in samples consisting of complete DMEM containing 10% of FBS (Dulbecco's Modified Eagle Medium + 10% Fetal Bovine Serum DMEM-FBS).
  • FBS Dulbecco's Modified Eagle Medium + 10% Fetal Bovine Serum DMEM-FBS
  • a standard solution PBS pH 7.4 containing a mixing of IBU (10 pg/mL) and T3 (20 pg/mL) were analysed.
  • the obtained chromatograms did not present additional peak at the retention time of IBU.
  • different samples consisting of complete DMEM DMEM + 10% FBS
  • samples of DMEM without FBS
  • the mass spectra of T3 was acquired after direct infusion of the tuning standard solutions. The higher response was obtained using an ESI operating in positive ionization.
  • T3 was identified on the basis of retention time, the presence of two specific mass transitions and ion ratio of these product ions.
  • the specificity was evaluated by checking the ion chromatograms of 10 blank samples of culture medium extracted and analyzed with the above-mentioned method for potential co-eluting interfering compounds, which can impair interpretation at the specific retention time of the analytes.
  • the linearity of the method was assessed with matrix-matched calibration curves freshly prepared each analysis day. The regression lines obtained were all satisfactory, with a coefficient of determination (r2) always higher than 0.98, in the range from 0 to 200 ng/mL.
  • the accuracy of the determination was assured by preparing and analyzing twice (at the beginning and at the end of each batch of analysis) the calibration curve in cell colture medium.
  • T3 content in PLLA fibers loaded with two drugs (IBU and T3) was determined in five samples.
  • the average content of T3 was l.73 ⁇ 0.4l pg/mg of fiber.
  • the fibers obtained using the PLGA 50:50-IBU conjugated showed a drug content of
  • micrometric and nanometric fibers (based on PLLA and containing 5% IBU) were produced. Both materials (micro and nano) showed a similar release profiles, however the difference in fiber size affected the amount of drug released. As expected, the percentage of the drug release from nanometer fibers was higher than from the microfibers (42% and 28% after 14 days, respectively) (FIG. 16).
  • the system based on 5% IBU loaded PLLA was selected for the production of electrospun fibers containing both IBU and T3.
  • the final system showed the desired controlled drug release profile, delivering the target amount of IBU in the prefixed time. T3 and IBU release in the electrospun fibers.
  • rat contusion model has been extensively characterized, and the most significant parameters for efficacy studies are presented in Fig. 7.
  • Basso-Beattie-Bresnahan 21 point score for evaluation of locomotor recovery after spinal lesion lesion group animals show a substantial decrease in locomotion after spinal cord injury, which is partially recovered over the time. Selected parameter of the gait analysis performed by CatWalk apparatus are presented in Fig. 13.
  • coordination parameters (Duty Cycle, Step Sequence Regularity Index) describing the ability of animals in coordinating movements indicates that injured animals show differences in all parameters analysed compared to sham animals. Notably, the duty cycle is recovered. The lesion volume has been also estimated, and results are presented in Fig. 17 Control treatment: systemic administration

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